title: Development of an HPLC method for measuring orally administered 1-substituted 2-alkyl-3-hydroxypyrid-4-one iron chelators in biological fluids.
abstract: "High-performance" liquid-chromatographic (HPLC) methods have been developed for identifying 1-substituted 2-alkyl-3-hydroxypyrid-4-one iron chelators in serum and urine. Ion pairing with heptane- or octanesulfonic acid in pH 2.0-2.2 phosphate buffer and reversed-phase chromatography were required to separate these compounds from endogenous compounds in both biological fluids. In both the 2-methyl and 2-ethyl series of 1-substituted compounds (H, methyl, ethyl, or propyl) the elution times increased in accordance with the n-octanol/water partition coefficients (propyl greater than ethyl greater than H greater than methyl). Urine samples were filtered (0.4 microns pore size) and injected either undiluted or after dilution with elution buffer. After the addition of internal standard, the plasma or serum samples were deproteinized by treatment with HCIO4, 0.5 mol/L, centrifuged, and the supernates were injected directly onto the HPLC. Using these procedures, we could identify 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in the serum and urine of a thalassemic patient who had received a 3-g dose of the drug and in the urine of other patients who had received the same dose. One or more possible metabolites were also observed in the chromatograms of both urine and serum. The 24-h urinary output of L1 (0.22-2.37 g) and iron (10.6-71.5 mg) varied but there was no correlation between the two with respect to quantity or concentration. Instead, urinary iron output was higher in patients with a greater number of transfused units of erythrocytes. This is the first study in humans to show that L1 is absorbed from the gut, enters the circulation, and is excreted in the urine.
authors.affiliation: Department of Haematology, Royal Free Hospital School of Medicine, London, U.K.
authors.forename: J G
authors.initials: JG
authors.lastname: Goddard
authors.forename: G J
authors.initials: GJ
authors.lastname: Kontoghiorghes
mesh_terms: D000293:Adolescent; D001803:Blood Transfusion; D001826:Body Fluids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D000077543:Deferiprone; D006801:Humans; D007501:Iron / Q000032:analysis* / Q000097:blood / Q000652:urine; D007502:Iron Chelating Agents / Q000032:analysis* / Q000627:therapeutic use; D008297:Male; D011728:Pyridones / Q000032:analysis* / Q000097:blood / Q000627:therapeutic use / Q000652:urine; D012997:Solvents; D013789:Thalassemia / Q000628:therapy
chemical_list: D007502:Iron Chelating Agents; D011728:Pyridones; D012997:Solvents; D000077543:Deferiprone; D007501:Iron